The controlled release of bioactive molecules from polymer matrices or polymer capsules has been proposed as a promising approach in various therapeutic interventions in order to avoid multiple dosing and to sustain continuous or pulsed release over time. The entrapment and immuno-isolation of small-molecule drugs, hormones, protein therapeutics or cell lines engineered for production of biologics in the patient's body have been designed for the treatment of various diseases such as infections, cancer, diabetes and different genetic disorders. Most controlled release systems currently available have either been chemically designed for sustained auto-catalytic or tissue-specific discharge of the therapeutic cargo, or engineered to release the therapeutic load in response to physical cues such as pH, light, ionic strength, magnetic resonance, or an electric field. Unfortunately, polymers designed for controlled release are often limited in their chemical flexibility, while most physical stimuli are impractical for in vivo applications. Also, the timing of release and overall release kinetics are often difficult to control.
Microencapsulation of viable genetically modified cells has become a widely used technology for cell-based therapeutic strategies and biopharmaceutical manufacturing. The encapsulation in biocompatible and immuno-isolating matrices protects the cells from environmental stress while providing favourable local conditions. Additionally, nutrients, waste products and therapeutics may freely penetrate the semi-permeable membrane of capsules. The ability to implant genetically engineered cells in immuno-protective materials may have great potential for therapeutic uses. The integration of mammalian cells in varying encapsulating polymers have lead to therapeutic strategies for the treatment of cancer, diabetes, hemophilia B, ischemia heart disease and other human disorders [see e.g. Zhang Y, Wang W, Zhou J, Yu W, Zhang X, Guo X, Ma X, Ann Biomed Eng 2007; 35:605-14]. In animal models, the immuno-protection by microcapsules even allowed the transplantation of xenogenic cells without rejection of implanted cells [Schneider S, Feilen P J, Brunnenmeier F, Minnemann T, Zimmermann H, Zimmermann U, Weber M M, Diabetes 2005; 54:687-93]. Additionally, a host-independent long-term drug delivery method by encapsulated cells has been reported in a mouse disease model [Orive G, de Castro M, Ponce S, Hernandez R M, Gascon A R, Bosch M, Alberch J, Pedraz J L, Mol Ther 2005; 12:283-9].
In the last three decades various materials were tested for their potential immuno-protection and biocompatibility properties. Sodium alginate, a natural polymer isolated from brown algae, which is able to precipitate in the presence of poly-L-lysine (PLL) was widely used for the production of microcapsules. However, the low quality reliability and poor biocompatibility of the precipitation agent PLL resulted in its replacement by other materials. In various studies sodium cellulose sulfate (CS)/poly-diallyl-dimethyl-ammonium chloride (pDADMAC) capsules showed less immunogenicity and higher biocompatibility than alginate/PLL. Furthermore, CS/pDADMAC capsules can be produced in a one-step high throughput procedure [Weber W, Rimann M, Schafroth T, Witschi U, Fussenegger M, J Biotechnol 2006; 123:155-63]. A clinical phase I/II long-term study demonstrated that CS/pDADMAC encapsulated cells showed no foreign body reaction or alteration of the recipient immune system and that cells may survive for a nearly unlimited time span [Gunzburg W, Salmons B, Trends Mol Med 2001; 7:30-7]. CS/pDADMAC encapsulated cells can be also successfully frozen and retain viability after thawing. Cellulases, which can cleave the polymer backbone of CS/pDADMAC capsules, are typically absent from mammalian tissues.
TET [Gossen M, Bujard H, Proceedings of the National Academy of Sciences USA 1992; 89(12):5547-51] or E.REX [Weber W, Fux C, Daoud-el Baba M, Keller B, Weber C C, Kramer B P, Heinzen C, Aubel D, Bailey J E, Fussenegger M, Nature biotechnology 2002; 20(9):901-7] are systems for trigger-inducible expression and secretion by mammalian cells. TET/E.REX are prototypic transgene control system which are responsive to clinically licensed antibiotics (tetracycline/doxycycline, erythromycin) and consist of chimeric transactivators designed by fusing bacterial response regulators to a eukaryotic transactivation domain, which binds and activates promoters containing transactivator-specific operator sites 5′ of minimal eukaryotic promoters. In the presence of regulating antibiotics the transactivators are released from their cognate promoters and transgene expression is silenced in a dose-dependent manner [Weber W, Fussenegger M, Current opinion in biotechnology 2007; 18(5):399-410; Weber W, Fussenegger M, The journal of gene medicine 2006; 8(5):535-56].
Artificial insemination (AI) of cattle is the major reproduction technology used in modern stock farming. In northern and western European countries the artificial inseminated reproduction of diary cattle exceeds 95%. The success of AI is strongly dependent on a precise determination of ovulation and a temporally coordinated insemination. The efficiency of AI is limited by the sperm survival in utero, which is compromised by leucocyte mediated phagocytosis and sperm retrograde transport, limiting the fertilization period to approximately 20 hours. As a result, the rate of successful artificially inseminated cows (non-return rate) does not exceed 70%. The ovulation in mammalian is a complex and primarily hormone-controlled process that plays a critical role in reproductive physiology. Initiation of ovulation is stimulated by a strong and highly specific preovulatory surge of the pituitary luteinizing hormone (LH). The LH binds to the luteinizing hormone receptor (LHR) that is expressed on the granulosa and theca cells of the mature preovulatory ovarian follicle. Upon activation the LHR couples to numerous G-proteins resulting in the stimulation of the cyclic adenosine monophosphate (cAMP) and inositol-phosphate signaling cascades followed by reprogramming of the cells. The luteinization of the granulosa and theca cells leads to a rupture of the mature follicle and a release of the fertilizable oocyte.